








key: cord-308080-1heu9vuv
authors: Simulundu, Edgar; Mupeta, Francis; Chanda-Kapata, Pascalina; Saasa, Ngonda; Changula, Katendi; Muleya, Walter; Chitanga, Simbarashe; Mwanza, Miniva; Simusika, Paul; Chambaro, Herman; Mubemba, Benjamin; Kajihara, Masahiro; Chanda, Duncan; Mulenga, Lloyd; Fwoloshi, Sombo; Shibemba, Aaron Lunda; Kapaya, Fred; Zulu, Paul; Musonda, Kunda; Monze, Mwaka; Sinyange, Nyambe; Liwewe, Mazyanga M.; Kapin’a, Muzala; Chipimo, Peter J.; Hamoonga, Raymond; Simwaba, Davie; Ngosa, William; Morales, Albertina N.; Kayeyi, Nkomba; Tembo, John; Bates, Mathew; Orba, Yasuko; Sawa, Hirofumi; Takada, Ayato; Nalubamba, King S.; Malama, Kennedy; Mukonka, Victor; Zumla, Alimuddin; Kapata, Nathan
title: First COVID-19 Case in Zambia – Comparative phylogenomic analyses of SARS-CoV-2 detected in African countries
date: 2020-10-06
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2020.09.1480
sha: 
doc_id: 308080
cord_uid: 1heu9vuv

Since its first discovery in December 2019 in Wuhan, China, COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread rapidly worldwide. Whilst African countries were relatively spared initially, the initial low incidence of COVID-19 cases was not sustained for long due to continuing travel links between China, Europe and Africa.. In preparation, Zambia had applied a multisectoral national epidemic disease surveillance and response system resulting in the identification of the first case within 48 hours of the individual entering the country by air travel from a trip to France. Contact tracing showed that SARS-CoV-2 infection was contained within the patient’s household, with no further spread to attending health care workers or community members. Phylogenomic analysis of the patient’s SARS-CoV-2 strain showed it belonged to lineage B.1.1., sharing the last common ancestor with SARS-CoV-2 strains recovered from South Africa. At the African continental level, our analysis showed that lineage B.1 and B.1.1 lineages appear to be predominant in Africa. Whole genome sequence analysis should be part of all surveillance and case detection activities in order to monitor the origin and evolution of SARS-CoV-2 lineages across Africa.

The WHO declared COVID-19, caused by SARS-CoV-2 a Public Health Emergency of International Concern (PHEIC) on 30 th January 2020 and later a Pandemic on 11 th March 2020. 1 As of 28 th September, 2020 there were 32.7 million COVID-19 cases with 991,000 deaths worldwide reported to the WHO. 2 All African countries have been affected and have reported a total of 1,172,342 COVID-19 cases including 25,481 deaths. 2 As the global COVID-19 events unfolded, and Africa's first COVID-19 case was reported from Egypt on 14 th February 2020, many African countries prepared for the arrival of COVID-19. 3 Zambia embarked on the intensification of the disease surveillance and emergency preparedness and response systems, including activating the Public Health Emergency Operations Centre (PHEOC). As part of preparedness activities, ports of entry were put on alert and thermal scanners were installed for screening incoming passengers at airports and ground crossing. Key to the preparedness was ensuring that local and international networks were functional, including staff training and were crucial for early detection of imported cases given that Zambia was at risk for importation of COVID-19. 4 The University Teaching Hospital (UTH) Virology Biosafety level-2 (BSL-2) Laboratory, and the University of Zambia School of Veterinary Medicine BSL-3 Laboratory in Lusaka, Zambia were identified as national COVID-19 diagnostic testing centers and for molecular analyses of SARS-CoV-2 lineages. We report the identification and clinical management of the first COVID-19 case from Zambia, and present the phylogenetic analyses of the patient's SARS-CoV-2 isolate, comparing it to other SARS-CoV-2 lineages reported from other African countries.

Ethical review and approval to publish: Ethical approval for case study and phylogenomic sequencing, and publication of this case study was obtained from the University of Zambia positive only in the case under study. Further questioning indicated that the patient had a slightly dry and sore throat. On 18 th March, three days after returning to Lusaka, our case developed a mild fever (38.0 o C) and was treated using paracetamol 1 gm three times a day orally for 5 days.

Similar to reports from Europe and the USA 5-7 patient had anosmia and complained of a metallic taste in the mouth two days prior to complete loss of taste. Our patient did not have any co-morbidities and was placed under quarantine for 21 days after testing positive to SARS-CoV-2. On day-5 (20 th March 2020), he developed a mild cough, persistent fever (>38 o C) , chest discomfort and clinical examination revealed bilateral chest crepitations. There were bilateral infiltrates on chest X-ray and full blood count showed mild lymphopenia (Table 1, Figure 1 ).

He was thus classified as having 'moderate COVID-19 pneumonia' and transferred for further clinical management at the national COVID-19 specific hospital where he was isolated, and was given a course of azithromycin (and then switched to amoxycillin-clavunate acid) and supportive care. Smell and taste abnormalities resolved within 5 days. The patient did not require intensive care and steadily improved with resolution of fever and resolution of infiltrates on repeat chest radiograph. He made a full recovery by day 21. Repeated contact tracing within the household, showed his wife testing positive for SARS-CoV-2, but his children remained negative. There was no further spread to other family members or attending health care workers.

Nasopharyngeal and oropharyngeal swab specimens were collected on 16 th March 2020 in accordance with the CDC recommendations, 8 and samples were processed using standard RT-PCR methodology for SARS-CoV-2 detection.

For whole genome sequencing, the Sanger method was employed using several overlapping primers designed using Geneious software version 10.0.9. The list of primers and their combinations for RT-PCR assays and sequencing are listed in Table S1 . The complete genome of SARS-CoV-2 investigated in this study was deposited in GenBank (accession no. MT790522). The whole SARS-CoV-2 genome generated in this study, together with other selected SARS-CoV-2 sequences accessed from GISAID data base were aligned using the FFT-NS-2 algorithm available in the multiple sequence alignment program (MAFFT) using default settings. 9 The selection included mostly representation from all African countries that had deposited whole genomes in the GISAID database as of 30 th August, 2020. The resulting final alignment was then uploaded to the IQ-TREE webserver 10 for construction of a Maximum Likelihood phylogeny using the general time reversible nucleotide substitution model and rate of heterogeneity set to gamma (GTR+G), otherwise default settings. Branch robustness was estimated using ultrafast bootstrapping tool available in IQ-TREE with 1000 replicates. 11 The ML tree was then rooted using TempEst (version 1.5.1), 12 which estimated the best-fitting root of this phylogeny using the heuristic residual mean squared function, aimed at minimizing the variance of root-to-tip distances. The resultant ML tree file was edited using iTOL. 13 Viral lineages were identified in the phylogeny according to the recently described nomenclature 14 as well as demonstrated recently in Uganda. 15 

Phylogenomic analysis showed that the detected SARS-CoV-2 belonged to lineage B.1.1, sharing the most common recent ancestor with viruses detected in South Africa (Figure 2) Wuhan-Hu-1, which included the D614G mutation which has been observed to correlate with increased case fatality rates. 16 The SARS-CoV-2 reported in this study also had the P4715L mutation which has been observed to occur in almost all strains with D614G mutation, which might affect the speed of virus replication. 17 SARS-CoV-2 variants with P4715L predominate in Europe and the USA. 17 Whilst most (if not all) complete genomes of SARS-CoV-2 deposited in public databases were sequenced using next generation sequencing (NGS) platforms, we utilized the Sanger method, which is more widely available in Africa when compared to NGS. As laboratory capacities for NGS are yet to be developed in many African countries, the Sanger method could still be used to sequence a number of complete genomes enough to provide a better understanding of the molecular epidemiology of SARS-CoV-2 in Zambia.

The epidemic preparedness and response in Zambia remains on high alert for the COVID-19 pandemic and capacity for contact tracing in the community has been built steadily over time due 

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ES, NK, AZ, JT, MB and AZ are members of The Pan-Africa-Europe Network for Emerging and Re-emerging Infections (PANDORA-ID-NET) -Website: https://www.pandora-id.net/partners).AZ is in receipt of a UK NIHR Senior Investigator Award, and 2020 Mahathir Science Award.We would like to thank all Laboratory staff from the National Influenza Center, Virology Laboratory, University Teaching Hospitals for technical assistance. Port health staff and health care workers involved in identification and managing our case, respectively, are duly acknowledged.J o u r n a l P r e -p r o o f

